Gene regulation is a fundamental process necessary for normal cell function, cell differentiation, and for responding to environmental stimuli. In addition, it plays a significant role in many important pathologies, such as cancer and viral infections. Regulation at the post-transcriptional level has become extremely important in cancer, linked with the early stages of tumorigenesis and with metastasis. On the subcellular level, it has been directly linked to mRNA localization and dynamics, mRNA-protein and mRNA-miRNA interactions. Currently, there have been no studies of native mRNA dynamics on the single RNA or granule level in living mammalian cells. The reason for this has been the lack of methods to image native RNAs on the single RNA or granule level. To address this issue, the Santangelo lab has developed a single RNA sensitive imaging strategy recently published in Nature Methods. Our methodology consists of two parts;the probes, which bind to native RNA via Watson-Crick pairing, and efficient cytosolic delivery via streptolysin O. Our probe design consists of four high-affinity, nuclease resistant, linear nucleic acids, labeled with multiple, high quantum-yield fluorophores linked together by streptavidin, via the biotin-streptavidin linkage, where streptavidin is the probe core. Target RNA is identified by the enhanced signal-to-background ratio achieved through binding of multiple probes per RNA. In this way, the identification of target is achieved in an analogous way to that of GFP-RNA binding protein or peptide systems, but native target sequences are utilized and significantly fewer binding sites. In preliminary experiments, probes bound rapidly to native, cytosolic target RNA (<10 minutes), and allowed for the first demonstrations of single RNA sensitivity, RNA granule trafficking, and dynamic RNA- protein colocalization studies, when used in conjunction with fluorescent fusion proteins. Therefore, the short term goal of this study is to fully validate our single molecule-sensitive imaging approach for the study of RNA trafficking, and quantify the dynamics of native 2-actin mRNA on the single RNA or granule level while trafficking to translation sites, a known regulatory process, highly associated with cell motility. We will also compare these results with those from plasmid-derived RNAs. Our long term goals are two-fold, 1) to develop the ability to use mRNA dynamics measurements to indirectly measure mRNA function, and 2) to utilize this imaging methodology in conjunction with siRNA, fluorescent antibody staining and fusion proteins, to determine the roles of trans-acting factors, such as RNA binding proteins and miRNAs, in the regulation of gene expression on the post-transcriptional level during a range of regulatory processes. This should enable new methods to intervene and control these processes, especially as applied to preventing cancer cell metastasis or making these cells more susceptible to treatment. PUBLIC HEALTH RELEVANCE: Gene regulation plays a critical role in many important biological problems and processes, such as cancer pathogenesis and stem cell differentiation, viral infections, and the assembly of neural circuits. Recently, RNA dynamics have been strongly linked to post-transcriptional gene regulation through the mechanisms of local translation and through the dynamic trafficking of mRNA between polysomes, P-bodies, and stress granules. In this grant we will fully validate a single molecule sensitive, fluorescent, native RNA imaging methodology, quantify the dynamics of native RNAs while trafficking to local translation sites, and compare these results with previous plasmid-derived RNA dynamics, in order to either confirm the use of plasmid-derived RNA as a model of native RNA dynamics or identify differences in motion, possibly due to variations in cis-acting sequences, RNA structure or as a consequence of traveling through a different biogenesis pathway.